1. Field of the Invention
This invention relates to inhibitors of influenza virus neuraminidase. In particular, this invention provides novel inhibitors to human influenza virus types A and B neuraminidase, methods of making the inhibitors, methods of treatment using the inhibitors and methods of prophylaxis from influenza infection.
2. Background of the Invention
Influenza virus epidemics occur every winter, causing significant morbidity and mortality in the U.S. population. Vaccines must be reformulated each year in response to antigenic variation and are frequently ineffective against new influenza variants. The only licensed anti-influenza drug, amantadine, and the related compound rimantadine, are effective only against influenza subtype A, and the virus can rapidly acquire resistance.
Influenza viruses are enveloped RNA viruses that are classified into three serological types: A, B, and C. Two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are responsible for the antigenic properties of the viruses. For influenza A, there are at least 9 subtypes of NA and 13 subtypes of HA, in contrast to only one subtype of influenza B NA and HA. Influenza C contains both HA and NA activities in a single surface glycoprotein, and only one subtype is known (Hay et. al., 1991 ).
Influenza C is not considered a serious disease and most adults have protective antibodies. Immunity from influenza A and B, however, does not last long since the virus is constantly undergoing antigenic variation. Influenza A undergoes a progressive antigenic drift and, in addition, undergoes an antigenic shift every 10-20 years in which a "new" HA and sometimes NA appears. In some cases, antigenic shift has resulted from the reassorting of gene segments from animal and bird viruses into a human strain. Influenza B has not undergone antigenic shift, perhaps because it is not found in birds or animals. Since 1977, there have been 3 influenza viruses circulating in humans: influenza A subtypes H3N2 and H1N1, and influenza B. Epidemics occur every winter, and usually one virus predominates. The current virus (c. 1993) has significantly different antigenic properties compared to the previously circulating variant or vaccine strain. The disease has a high infection rate and costs to the U.S. in a bad year are estimated to be 3-5 billion dollars (Murphy and Webster, 1990). The elderly are at high risk for serious complications from influenza, and excess mortality in the U.S. is estimated to be 10,000-20,000 each winter. Currently available vaccines and drugs have clearly failed to control influenza in humans.
The trivalent influenza vaccine currently licensed in the U.S. contains formalin-inactivated whole virus or partially-purified HA and NA (split vaccine) from H3N2, H1N1, and B strains. The whole virus preparation is more antigenic than the split form, but its toxicity precludes administration to young children. Neither vaccine confers long term protection, and even in the absence of antigenic variation, the vaccine would have to be given every year. Clinical trials of the promising cold-adapted live attenuated vaccine have not yet shown increased efficacy (Wright, 1992), although toxicity in young children is reduced compared to the split vaccine (Edwards et. al., 1991). The elderly, who are at greatest risk for severe complications and death from influenza, react poorly to the killed vaccine and have shown no better response in clinical trials with the live, attenuated vaccine (Powers et. al., 1991).
Only two anti-influenza drugs, amantadine and rimantadine, are currently licensed in the United States. They act by blocking the viral-coded ion channel (M2 protein) in influenza A but have no effect on influenza B, which uses a different ion channel. Resistance to amantadine or rimantadine develops quickly; typically the primary patient benefits from amantadine therapy, but contracts a resistant virus (Hayden and Hay, 1992). Therefore, a need exists for new, broad-spectrum anti-influenza drugs that act by different mechanisms.
The two major surface glycoproteins of influenza viruses A and B are essential for infectivity and offer potential targets for antiviral drug development. Hemagglutinin (HA) is responsible for viral attachment to host cells by binding to terminal sialic acid residues on host cell surface glycoconjugates, and HA is also involved in mediating membrane fusion. Neuraminidase (NA) (also called sialidase or acyineuraminyl hydrolase, EC 3.2.1.18) destroys the host cell viral receptor by catalyzing the hydrolysis of .alpha.-2,3- or .alpha.-2,6-glycosidic bonds to terminal sialic acid residues of surface glycoconjugates (Paulson, 1985; Daniels et. al., 1987; Suzuki et. al., 1986). This facilitates release and prevents aggregation of progeny virus (Palese et. al., 1974). Therefore, the inhibition of either coat glycoprotein is desirable and should provide antiviral effects. In particular, the structure-based design of inhibitors for NA is highly desirable.
Effective inhibitors of NA thus should provide anti-influenza agents. For instance, monoclonal antibodies against NA were shown to terminate viral infection, and the anti-NA response was protective (Webster et. al, 1988). A neuraminidase-minus mutant of influenza A (Liu and Air, 1993) was produced, and the mutant was unable to replicate more than one cycle without added exogenous neuraminidase. Additionally, mutant influenza A viruses lacking the NA stalk do not replicate in eggs and are much less virulent in mice (Castrucci and Kawaoka, 1993).
Influenza NA, which accounts for 5-10% of the virus protein, has an approximate molecular weight (MW) of 250,000 and lies mostly outside of the viral membrane. It is a tetramer with C4 symmetry and consists of an N-terminal membrane-anchored domain, a stalk, and a globular head. Each subunit of the head contains a catalytic site and is a glycosylated polypepfide with MW 50,000 containing 6 .beta.-sheets arranged in a propeller formation. Protcolytic cleavage of the stalk has produced biologically and antigenically active heads (Air and Laver, 1989). Heads from several viruses have been crystallized, and x-ray structures of NA heads from influenza A N2 (Varghese et. al, 1992), N9 (Bossart-Whitaker et. al., 1993; Tulip et. al., 1991), N9 complexed with monoclonal antibody NC41 (Tulip et. al., 1992), and two B virus NA's (Janakiraman et. al, 1994; Burmeister et. al, 1992) have been solved. These studies reveal that, while sequence homology among neuraminidases is often low (influenza A N9 NA and B/Lee NA have only 28% sequence homology), the tertiary structures are well-conserved. In particular, a group of 18 conserved amino acid residues constitute a strain-invariant sialic acid binding site among all influenza NA's thus far studied.
Thus, effective inhibitors of NA will provide highly desirable anti-influenza agents against type A and B variants. Site-specific mutations of conserved residues provided correctly folded mutants that, in most cases, were enzymatically inactive (Lentz et. al, 1987). The apparent importance of this conserved site to viral replication suggests that the virus may not be able to evade anti-influenza NA inhibitors through mutation. Additionally, since NA acts at the end of the viral replication cycle, a potential advantage of anti-influenza NA inhibitors is that sufficient viral protein may be provided to stimulate the immune system.
The prior art lacked available atomic coordinates for earlier NA structures. Thus, the x-ray crystal structures for NA first had to be solved. In this fashion, original structures for an N9 influenza A NA (Bossart-Whitaker et. al, 1993) and an influenza B NA (Janakiraman et. al, 1993) were obtained. These have been refined as both the native enzyme and as complexes with several ligands, including sialic acid and DANA (discussed infra). Also, crystals have been obtained and the x-ray structure refined-for a published N2 influenza A NA.
Influenza NA exhibits a broad pH range with the optimum between 5.8 and 6.6, and using a small trisaccharide substrate (N-acetyl neuraminyl lactose), the K.sub.m is about 0.4 mM (Drzeniek, 1972; Mountford et. al., 1982). The product of catalysis, sialic acid (N-acetylneuraminic acid or NANA) is a modest inhibitor with a K.sub.i of about 1 mM. A number of other compounds have been evaluated as in vitro inhibitors of influenza neuraminidase, and among the most potent thus far described is 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), which inhibits with a K.sub.i of 10 .mu.M (Meindl et. al., 1974). A DANA analog, 2-deoxy-2,3-dehydro-N-(trifiuoroacetyl)neuraminic acid (FANA), exhibited greater in vitro activity (Palese and Compans, 1976). While no reports have described in vivo anti-influenza effects for either DANA or FANA, several new DANA analogs have recently been described that possess in vivo antiviral effects, confirming that inhibitors of NA provide useful anti-influenza agents (Von Itzstein et. al., 1991; O'Neill, 1993).
The 20 year old observation that DANA was an effective in vitro inhibitor of NA has resulted in the preparation of a large number of synthetic derivatives varied mainly at the 2-, 4-, 5-, and 6-positions (for example, Meindl et. al, 1974; Schreiner et. al., 1991; Kumar et al., 1982; Vasella et. al., 1991). Unfortunately, except for an early study (Meindl et. al., 1974), these have not been assayed with influenza NA, but instead with NA from Vibrio cholerae or Arthrobacter sialophilus. Numerous synthetic sialic acid (NANA) analogs (for example, Glanzer et al., 1991; Yamamoto et al., 1992; Mack et al., 1992) have also been reported, although these are typically much less effective inhibitors of NA than DANA or FANA. It has been suggested that DANA, which unlike NANA contains a double bond at the C2-C3 position, is a planar "transition state analog" inhibitor (Flasher et al., 1983), since it may mimic a planar oxonium cation intermediate suggested to be involved during hydrolysis. Recent studies of influenza virus NA using NMR, molecular dynamics, and kinetic isotope effects support a sialosyl cation transition-state complex in the reaction (Chong et. al., 1992). Finally, only a few novel NA inhibitors that are not pyrans or furans have been described, including isoquinolines (Brammer et al., 1968), .alpha.-mercaptocinnamic acids and imidazoles (Haskell et. al., 1970), oxamic acids (Brossmer et. al., 1977), the piperidine, siastatin B, and derivatives (Kudo et. al., 1993), and plant flavonoids (Nagai et. al., 1992). Due to limited investigations with influenza virus NA, and the possibility that NA's from such diverse sources as viruses and bacteria have different binding sites (even Vibrio cholerae and Arthrobacter sialophilus show different inhibitor specificity) (Wang et. al., 1978; Miller et. al., 1978), these previous structure-activity relationship (SAR) studies provide little reliable assistance for designing new anti-influenza drugs. It is clear that a novel approach to the design of new NA inhibitors is needed.
One attempt at rational design of inhibitors has resulted in a class of compounds based on 2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en). These inhibitors have K.sub.i binding constants as high as 10.sup.-10 M, but they are excreted from-the infected person's body very rapidly and thus are not efficient. Oral activity has not been reported. (Brammer, Nature, 1968).
It should be noted that, for therapeutic utility as anti-influenza agents, inhibitors of influenza NA should not inhibit mammalian sialidases. The latter have been implicated in a number of important metabolic processes including the regulation of cell proliferation (Usuki et. al., 1988a), the clearance of plasma proteins (Ashwell and Morell, 1974), and the catabolism of gangliosides and glycoproteins (Usuki et. al., 1988b). The optimization of binding to influenza NA described in the structure-based approach proposed herein will provide selective inhibitors and concomitant methods for making and using them.